The major aim of this proposal is to investigate a novel regulatory mechanism that may contribute to gene expression differences between individuals. MicroRNAs anneal to the 3'UTR of mRNAs and typically negatively regulate gene expression. We hypothesize that microRNAs regulate the expression of the serotonin receptor 1B (HTR1B) and that variation in the microRNA response elements will modify HTR1B gene expression and as a consequence may alter behavior and potentially risk for alcohol dependence. Utilizing a luciferase assay we have found that a moderately common variant in the 3'UTR of the HTR1B mRNA disrupts a critical base pairing required for the activity of the microRNA miR-96, thus this variant moderates miR-96 regulation and gene expression. Male transgenic mice that lack HTR1B are more aggressive than their male littermates;therefore we hypothesized that individuals who carry the G-element, which attenuates miR-96 mediated repression, may display different aggression-related behaviors. In our initial studies at the University of Connecticut Health Center Alcohol Research Center, we have found that male and female university students, who carry the G-element, endorse fewer conduct disorder (CD) items. In further analysis, we found that for male students, carriers of the G-element report less hostile mood states. Thus it appears that this functional variant may alter the expression of HTR1B and potentially manifests in differences in human behavior. Understanding this regulatory phenomenon and how gene expression differences manifest will likely contribute to our knowledge of the genetics underlying alcohol dependence. Therefore Aim 1 of this proposal will investigate whether the polymorphism that moderates regulation of miR-96 in the luciferase assay is associated with HTR1B gene expression differences in human lymphoblastoid cell lines. In Aim 2 we will use an in-vitro luciferase assay to explore the nucleotide structure required for the regulation of the HTR1B response element. The final aim will evaluate whether polymorphism in putative microRNA binding sites that have been identified in-silico in other candidate genes relevant to alcohol use disorders (including CRHR1, CNR1, DRD2, COMT, HTR2A, HTR2C, OPRM1, PDYN, GABRA6, MANEA) also modify microRNA regulation. In sum, the proposed aims will explore a new regulatory mechanism of HTR1B expression, and the proposed experiments will prove invaluable in understanding microRNA regulation of HTR1B and other candidate genes related to the vulnerability for alcohol-related problems, including alcohol dependence. This proposal will also explore the potential functional variants in microRNA target sites of other candidate genes.